A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Development and validation of a reverse phase liquid chromatography method for the simultaneous quantification of eserine and pralidoxime chloride in drugs-in-adhesive matrix type transdermal patches.

BACKGROUND: In the present study, a simple, precise, specific, fast, accurate and reliable reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous determination and quantification of eserine and pralidoxime chloride in drugs-in-adhesive matrix type transdermal patches. METHODS: The chromatographic separation was achieved by C18 column, using a mobile phase consisting of acetonitrile: 10 mM potassium dihydrogen phosphate, 10 mM heptane-1-sulfonic acid sodium salt monohydrate in water (30:70, v/v) adjusted at pH 3.0 with ortho-phosphoric acid. Flow rate was 1.0 mL/min and UV detection at 238 nm. The method was validated according to the International Conference on Harmonization (ICH) guidelines. RESULTS: The calibration curves were linear over the different concentration ranges of 0.5-10 µg/ml for eserine and 5-25 µg/mL for 2PAM. Relative standard deviation for precision was less than 2.0%. Limit of detection values of eserine and 2-PAM were 0.018 µg/mL and 0.008 µg/mL, respectively. The limit of quantification of eserine and 2-PAM were 0.055 µg/mL and 0.026 µg/mL, respectively. CONCLUSION: The developed method was applied for the routine analysis of these 2 drugs in drugs-in-adhesive matrix type transdermal patches in order to evaluate the drug content of different formulations. It could be also used with reliability for the determination of the drug in other pharmaceutical dosage forms.

Stripping voltammetric determination of pyridine-2-aldoxime methochloride at the iron(III) doped zeolite modified glassy carbon electrode.

An iron(III) doped zeolite modified glassy carbon electrode was constructed for the determination of pyridine-2-aldoxime methochloride. X-ray diffraction and chemical analysis were utilized to determine the optimum pH and chemical content for doping zeolite. Cyclic voltammetry was used to characterize the modified electrode and study the kinetics of the acid treated and untreated modified electrode. Acid treatment of the modified electrode showed a better electrochemical behavior compared to the untreated iron(III) doped zeolite modified electrode. Square wave anodic stripping voltammetry was employed to investigate the working pH and preconcentration time. The analytical performance of the modified electrode was evaluated, and a linear anodic stripping response for pyridine-2-aldoxime methochloride in the concentration range of 0.5-100.0 µM with a detection limit of 1.61 × 10(-7) M was obtained. Finally, the developed method was successfully applied for the determination of pyridine-2-aldoxime methochloride in a biological sample.

Review of UV spectroscopic, chromatographic, and electrophoretic methods for the cholinesterase reactivating antidote pralidoxime (2-PAM).

Pralidoxime (2-PAM) belongs to the class of monopyridinium oximes with reactivating potency on cholinesterases inhibited by phosphylating organophosphorus compounds (OPC), for example, pesticides and nerve agents. 2-PAM represents an established antidote for the therapy of anticholinesterase poisoning since the late 1950s. Quite high therapeutic concentrations in human plasma (about 13 µg/ml) lead to concentrations in urine being about 100 times higher allowing the use of less sensitive analytical techniques that were used especially in the early years after 2-PAM was introduced. In this time (mid-1950s until the end of the 1970s) 2-PAM was most often analyzed by either paper chromatography or simple UV spectroscopic techniques omitting any sample separation step. These methods were displaced completely after the establishment of column liquid chromatography in the early 1980s. Since then, diverse techniques including cation exchange, size-exclusion, reversed-phase, and ligand-exchange chromatography have been introduced. Today, the most popular method for 2-PAM quantification is ion pair chromatography often combined with UV detection representing more than 50% of all column chromatographic procedures published. Furthermore, electrophoretic approaches by paper and capillary zone electrophoresis have been successfully used but are seldom applied. This review provides a commentary and exhaustive summary of analytical techniques applied to detect 2-PAM in pharmaceutical formulations and biological samples to characterize stability and pharmacokinetics as well as decomposition and biotransformation products. Separation techniques as well as diverse detectors are discussed in appropriate detail allowing comparison of individual preferences and limitations. In addition, novel data on mass spectrometric fragmentation of 2-PAM are provided.

Development and validation of a sensitive HPLC method for the quantification of HI-6 in guinea pig plasma and evaluated in domestic swine.

A rapid and small volume assay to quantify HI-6 in plasma was developed to further the development and licensing of an intravenous formulation of HI-6. The objective of this method was to develop a sensitive and rapid assay that clearly resolved HI-6 and an internal standard in saline and plasma matrices. A fully validated method using ion-pair HPLC and 2-PAM as the internal standard fulfilled these requirements. Small plasma samples of 35 microL were extracted using acidification, filtration and neutralization. Linearity was shown for over 4 microg/mL to 1mg/mL with accuracy and precision within 6% relative error at the lower limit of detection. This method was utilized in the pharmacokinetic analysis HI-6 dichloride (2Cl) and HI-6 dimethane sulfonate (DMS) in anaesthetized guinea pigs and domestic swine following an intravenous bolus administration. From the resultant pharmacokinetic parameters a target plasma concentration of 100 microM was established and maintained in guinea pigs receiving an intravenous infusion. This validated method allows for the analysis of low volume samples, increased sample numbers and is applicable to the determination of pharmacokinetic profiles and parameters.

Liquid chromatography-tandem mass spectrometry method for determination of the pyridinium aldoxime 4-PAO in brain, liver, lung, and kidney.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and applied to the in vitro determination of 4-[(hydroxyimino)methyl]-1-octylpyridinium cation (4-PAO), which can penetrate the blood-brain barrier and reactivate acetylcholinesterase (AChE) inhibited by alkylphosphonate in the brain, liver, lung, and kidney. The limit of detection (LOD) was 0.235 microg cation/g wet weight, and the quantification range and linearity of the calibration curve extended over a range of 0.470-941 microg cation/g wet weight. For the proof of applicability, when 4-PAO was administrated intravenously via the rat tail vein at 10% LD(50), we were able to quantify the 4-PAO concentration in the tissues: brain 7.60+/-1.32 microg cation/g wet weight (mean+/-SD, n=5), liver 26.8+/-2.82 microg cation/g, lung 76.4+/-24.9mugcation/g, and kidney 638+/-266 microg cation/g. In addition, the methods for 4-[(hydroxyimino)methyl]-1-decylpyridinium bromide (4-PAD) and 4-[(hydroxyimino)methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) were partly validated referring to the findings of the 4-PAO full validation. Thus, the LC-MS/MS method described in this study can be useful for quantification of pyridinium aldoxime methiodide (PAM)-type oximes in biological samples.

Analysis of pralidoxime in serum, brain and CSF of rats.

After administration of various amounts of pralidoxime to rats, the levels in serum, brain and cerebrospinal fluid (CSF) were measured using capillary zone electrophoresis (CZE). The calibration curves were established using spiked samples. The calibration covers the ranges from 0.3 - 200 microg/mL, 0.3 - 7 microg/mL and 0.1 - 7 microg/mL for serum, brain and CSF, respectively. The CZE measurement opens the way to the fast and reliable determination of pyridinium aldoxime concentrations in serum, cerebrospinal fluid and brain, thereby monitoring blood-brain and blood-CSF penetration of pyridinium aldoxime-type antidotes clinically used in organophosphate poisoning.

Simultaneous determination of hydroxylamine and cyanide in formulations containing pralidoxime salts by flow injection.

A flow injection method is described for the simultaneous determination of cyanide and hydroxylamine which are known decomposition products of formulations containing pralidoxime salts used in the treatment of anticholinesterase poisoning. By using the diffusion of HCN from the carrier stream followed by amperometric detection, high selectivity and sensitivity and a wide dynamic range can be achieved. Hydroxylamine is determined by its oxidation with iodine to nitrite which can then be determined colorimetrically. The gas diffusion unit effectively acts as a stream splitter for the two analytes allowing their simultaneous determination from a single sample injection. The performance of the system and its applicability to thermally stressed pralidoxime solutions are described.

Potentiometric investigation of the stability of palladium(II) complex of pralidoxime chloride in aqueous solution.

The formation of a complex between palladium(II) chloride and pralidoxime chloride (PAM-2Cl) has been studied by means of potentiometric pH measurements. The real stability constant of the complex in aqeous medium of ionic strength 0.3 M (KCl) at 25.0 degrees C was log Ks = 7.29. This value was close to that (log Ks = 7.02) obtained previously by spectrophotometric methods after appropriate correction with respect to the corresponding value of the acidic constant of PAM-2Cl (pKca = 8.05), which was also determined under the same experimental conditions.

Pralidoxime chloride stability-indicating assay and analysis of solution samples stored at room temperature for ten years.

A high-performance liquid chromatographic (HPLC) method is described for quantitation of pralidoxime chloride and its decomposition products 2-carboxy-, 2-formyl-, and 2-(aminocarbonyl)-1-methylpyridinium chloride. These decomposition products and 2-cyano- and 2-(hydroxymethyl)-1-methylpyridinium chloride and 1-methyl-2(1H)-pyridinone were separated from pralidoxime chloride on a silica gel column using a mobile phase of acetonitrile:water (86:14) in which the aqueous component was 8.36 mM in tetraethylammonium chloride and 52.5 mM in acetic acid. This method allows quantitation of the relatively low levels of 2-formyl-1-methylpyridinium chloride formed in acidic solution at room temperature. Sensitivity was shown to be at least 5 ng of the pralidoxime chloride and 15 ng of the 2-carboxy-, 2-formyl-, and 2-(aminocarbonyl)-1-methylpyridinium chloride injected on column. The coefficient of variation was 4% or less for all components measured. Autoinjectors containing 300 mg/mL of pralidoxime chloride in water were stored at room temperature for 8-10 years, followed by analysis for hydrogen cyanide using an ion-selective electrode. Less than 15 micrograms of cyanide per autoinjector was detected. The HPLC analysis of the solutions after being stored an additional 3-4 years at approximately 5 degrees C demonstrated that greater than 90% of the total of all measured components consisted of pralidoxime chloride. The remaining percentage was made up of 2-carboxy-, 2-formyl-, and 2-(aminocarbonyl)-1-methylpyridinium chloride.

Analysis of formulations containing pralidoxime mesylate by liquid chromatography.

An improved liquid chromatography procedure has been devised for the analysis of pralidoxime mesylate (P2S) and its degradation products in solution. An additional degradation product, 2-hydroxyaminoiminomethyl-1-methylpyridinium, has been positively identified and its route of formation and that of another recently identified product, 2-hydroxymethyl-1-methylpyridinium, has been established. Previous problems with the chromatography of 2-formyl-1-methylpyridinium have been resolved and the new method will resolve P2S and eight of its degradation products.

Stability studies of bis(pyridiniumaldoxime) reactivators of organophosphate-inhibited acetylcholinesterase.

Relative stability studies of three organophosphate-inhibited acetylcholinesterase reactivators, 1-(2-hydroximinomethyl-1-pyridinium)-3-(4-carbamoyl-1-pyridinium)- 2-oxapropane dichloride (HI-6), 1,1'-methylenebis(4-hydroximinomethylpyridinium) dichloride (MMB-4), and 1,1'-trimethylenebis(4-hydroximinomethylpyridinium) dibromide (TMB-4) were carried out by semiquantitative TLC and NMR methods. TMB-4 appears to be the most, and HI-6 the least stable of the three compounds. The extent of hydrolysis of HI-6, MMB-4, and TMB-4 in 0.05 M, pH 7 phosphate buffer was approximately 50, 25, and less than 1%, respectively, after 20 d at room temperature. The hydrolysis products of HI-6 were identified by NMR and MS (electron impact) as 2-pyridinealdoxime, picolinamide, and isonicotinamide, whereas that of MMB-4 was identified as 4-pyridinealdoxime. The stability of these reactivators decreases with increasing pH. TMB-4 was stable under both neutral and basic conditions at room temperature. Deuterium exchange of the methylene protons of MMB-4 in D2O and of the protons at the 2- and 6-positions of the pyridinium ring of TMB-4 in NaOD/D2O were observed.

Degradation pathway of pralidoxime chloride in concentrated acidic solution.

The degradation products of pralidoxime chloride (1) (X- = Cl-) in concentrated aqueous solutions (less than or equal to 50% w/v) were identified using one or more methods: HPLC, polarography, voltammetry, MS and/or NMR. The products found were the 2-cyano-, 2-carboxamido- and 2-carboxy-1-methyl-pyridinium chlorides, 1-methyl pyridinium chloride, cyanide ion, ammonia and carbon dioxide. 1-Methyl-2-pyridone was indirectly identified by the presence of cyanide ion. The degradation rate increased with increasing pH values between pH 1 and 3.2 and with increasing concentrations between 1 and 50% w/v pralidoxime chloride. The results suggest that 1 (X- = Cl-) is dehydrated by a hydroxyl-ion catalyzed reaction ot the nitrile 2 which is hydrolyzed to either the pyridone 6 and cyanide ion or to 2-carboxamido-1-methyl-pyridinium chloride 3. The amide is hydrolyzed to give the 2-carboxy derivative 4 which finally is decarboxylated to give 1-methylpyridinium chloride 5.

High-performance liquid chromatographic determination of pralidoxime chloride and its major decomposition products in injectable solutions.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pralidoxime chloride (I) and its major decomposition products in an injectable formulation is described. I and its decomposition products were detected and quantitated by their UV absorbances at 270 nm, after being separated from related compounds and formulation excipients on a reverse-phase C-18 column using a mobile phase consisting of 52% acetonitrile and 48% of an aqueous solution containing 0.005 M phosphoric acid and 0.001 M tetraethylammonium chloride. The major decomposition products of I in the injectable formulation were identified by their retention times and stop-flow spectroscopy as 2-carboxy-N-methylpyridinium chloride, N-methyl-2-pyridone, 2-carbamoyl-N-methylpyridinium chloride, 2-hydroxymethyl-N-methylpyridinium chloride, and 2-cyano-N-methylpyridinium chloride. A substance of unknown identity also was detected in degraded solutions of I. Stop-flow spectroscopy, employing the spectral discrimination technique, showed that the method is specific for I. Recovery of I from a spiked placebo formulation averaged 99.9%. The accuracy of the method was also demonstrated for the decomposition products over a range of concentrations representing 1-50% decomposition. Replicate determinations of I in degraded solutions gave coefficients of variation of 1.0 and 1.5%, while the precision of determining the decomposition products range from 1.3 to 6.5%. Regression lines with correlation coefficients greater than 0.9999 were obtained for I and its decomposition products, and solutions of these compounds were shown to be stable in the mobile phase for several days. Results for I by the HPLC and USP procedures are compared.

Ion-pair high-performance liquid chromatographic separation of a multicomponent anticholinergic drug formulation.

N,N'-Trimethylene-bis-(pyridinium-4-aldoxime)dibromide, 4-pyridine aldoxime, atropine sulfate, benactyzine hydrochloride, methyl paraben and propyl paraben are separated by ion-pair high-performance liquid chromatography. The method is specific for detecting and quantifying each compound in a complex mixture without solvent extraction or pretreatment. Levels as low as 1 ng on column are quantifiable by the procedure. All components are eluted within 9 min subsequent to the initial injection. Because of the simplicity of the method, the procedure is suitable for routinely monitoring the stability of the various compounds in the formulation during storage.

Ion-exclusion chromatography of compounds of biological interest.

The principle of ion exclusion was examined as a method for the separation of small ionic compounds. The systems employed consisted of very porous column packings, substituted with fixed charges, which were eluted by buffer solutions of low ionic strength. DEAE-Sephadex A-50 was principally employed, and it was shown that there was a linear relationship between the net charge on a cation and its partition coefficient into the gel phase. A similar relationship existed in the chromatography of amino acids on various columns bearing fixed negative charges. It was concluded that this was an efficient form of chromatography, which gave results directly related to the ionic charge of the sample being examined. The charge characteristics of biologically active compound could be determined by this method.